Identification of fetal dna and fetal cell markers in maternal plasma or serum

ABSTRACT

The present invention relates to the identification of fetal specific nucleic acids and fetal cell markers in maternal plasma or serum. In particular, the present invention relates to methods which rely on the analysis of polymorphic alleles of a population to determine an allele which is possessed by the fetus but absent from the mother. Fetal specific alleles identified using the methods of the invention can be used to quantify fetal DNA from maternal plasma or serum. In addition, antigens encoded by alleles identified using the methods of the invention can be targeted in methods of isolating or detecting fetal cells.

FIELD OF THE INVENTION

The present invention relates to the identification of fetal specificnucleic acids and fetal cell markers. In particular, the presentinvention relates to the identification of alleles of fetal DNA whichare not present in the genome of the mother. Fetal specific allelesidentified using the methods of the invention can be used to quantifyfetal DNA in a sample comprising fetal and maternal nucleic acids. Inaddition, antigens encoded by alleles identified using the methods ofthe invention can be targeted in methods of isolating or detecting fetalcells.

BACKGROUND OF THE INVENTION

Currently, fetal diagnosis is generally carried out by a procedure knownas amniocentesis which involves the aspiration of a small sample ofamniotic fluid from the pregnant mother, culturing the fetal cells inthe fluid, and determining the karyotype of the fetal cells. Recently,chorionic villus sampling has also been used, which involves the directtranscervical and transabdominal aspiration of the chorionic villus.However, as both amniocentesis and chorionic villus sampling requireinvasive procedures for obtaining fetal cells, they inevitably exposeboth the mother and the fetus to a certain amount of risk. Accordinglynon-invasive approaches to prenatal diagnosis are preferred.

Cell-free fetal DNA circulates in the plasma of pregnant women (Lo etal., 1997). Up to at least 7% of all cell-free DNA in maternalplasma/serum has been found to be of fetal origin. It has beendemonstrated that this fetal DNA can be used to predict the gender ofthe fetus (Lo et al., 1997), as well as to determine the fetal Rhesus Dstatus (Lo et al., 1998).

Studies have also indicated that the amount of fetal DNA in maternalplasma/serum can be correlated with certain fetal abnormalities such astrisomy 21 (Down syndrome) (Lee et al., 2002) and trisomy 13 (Wataganaraet al., 2003). Thus far, the quantitation of fetal DNA has generallyrelied upon Y-chromosome-specific sequences, which cannot serve tomeasure the DNA of female fetuses. Accordingly, a gender-independentquantitation of fetal DNA is needed.

There is a need for further methods which identify fetal specificnucleic acids and fetal cell specific markers. In particular, markersthat are gender independent are required which can be detected throughnon-invasive procedures.

SUMMARY OF THE INVENTION

The present inventor has devised methods for identifying genderindependent markers of fetal specific nucleic acids and fetal cells in apregnant female. These methods rely on the analysis of polymorphicalleles of a population to determine an allele which is possessed by thefetus but absent from the mother. These methods can be performed in anon-invasive manner through the analysis of samples comprising fetal andmaternal nucleic acids obtained from the pregnant female.

In a first aspect, the present invention provides a method ofidentifying an allele of a fetal cell, said allele not being present inmaternal cells, the method comprising;

-   -   i) identifying alleles which are not present in maternal cells,    -   ii) obtaining a sample from the mother comprising fetal and        maternal nucleic acids, and    -   iii) screening nucleic acids from the sample for at least one        allele not present in the maternal cells.

Steps i) and ii) can be performed in any order.

The general principle of the method of the first aspect can be appliedby typing all alleles at a particular locus, whether derived from thefetus or the mother. Thus, in a second aspect the present inventionprovides a method of identifying an allele of a fetal cell, said allelenot being present in maternal cells, the method comprising;

i) obtaining a sample from the mother comprising fetal and maternalnucleic acids,

ii) typing at least one locus from the sample obtained in step i),

iii) comparing the alleles identified in step with alleles of the samelocus possessed by the mother, and

iv) selecting an allele typed in step iii) which is not possessed by themother.

Any allele typed in step of the second aspect which is not possessed bythe mother will be a fetal marker inherited from the father. The methodof the second aspect differs from that of the first aspect, as the firstrelies on initially identifying alleles that, if possessed by the fetus,would be fetal specific followed by screening the sample for suchpotential fetal specific alleles, whereas the second aspect examines allalleles in nucleic acids obtained from the sample at a given locus (bothfetal and maternally derived) followed by excluding those which arefound in the mother to identify a fetal marker.

Step ii) of the second aspect may identify from the sample up to threealleles at a given locus, two from the maternal nucleic acids and onefrom the fetal nucleic acids which has been inherited from the father.Naturally, the fetal nucleic acids will comprise a second allele whichhas been inherited by the mother, however, it will typically not bedistinguished (unless a mutation has occurred) from that of the mother.More specifically, a primer amplifying a specific maternal allele, whichwas also inherited by the fetus, will amplify a single product from twosources, namely the maternal genome and the fetal genome. At the otherend of the scale the father and mother may be homozygous for the sameallele, and step ii) will only identify a single allele. If such a casearises, or if the fetus has inherited an allele from the father which isalso possessed by the mother, other loci will need to be examined toidentify a fetal marker. Preferably, many loci are typed in a singlereaction so that if one locus cannot be used as a marker the method willidentify a marker at another locus.

An important advantage of the above methods is that they do not requirethat the genotype of the father at the allele be known or determined.Accordingly, in a particularly preferred embodiment, the father is nottyped for any alleles.

In instances where the father is known, the methods may further comprisetyping the father at the same locus/loci as the mother, and identifyingalleles which are not present in the mother but which are present in thefather. Although the combination of the maternal and paternal typingwill provide a list of alleles which may be present in fetal DNA, if thefather is heterozygous at a given locus the maternal sample comprisingfetal and maternal nucleic acids will still need to be screened forfetal specific alleles to unequivocally identify at least some fetalmarkers as it cannot be predicted which one of the heterozygous alleleswould have been inherited by the fetus.

The methods can be performed at any stage during pregnancy. However, ina preferred embodiment, the sample is obtained during the firsttrimester of pregnancy.

The present invention relies on the genotype of the mother at the allelebeing investigated being known. Such alleles may have previously beencharacterized for other reasons, such as if it is the mothers secondchild and the method of the first or second aspect was performed duringher first pregnancy, or the mother has had an organ transplantation andhence the mother was HLA typed. However, in many cases the genotype ofthe mother of the allele(s) being investigated will be unknown. In suchcircumstances, the methods will further comprise typing alleles of atleast one locus of the mother. Naturally, the typing of the mother willat least target the same locus being investigated in the methods of thefirst and second aspects. With regard to the first aspect, the maternaltyping will be performed before step i).

The mother can be typed using any technique known in the art. Thisprocedure can be performed at a single locus or a number of loci. Forinstance, the typing can be performed by obtaining a genomic DNA samplefrom the mother and exposing the DNA to amplification and/or sequencingprocedure. In another example, the typing is performed by usingantibodies which bind the protein products of specific alleles.Preferably, the locus (loci) that are analysed encode proteins(antigens) that can be found on the cell surface and can bind antibodiesdirected against the protein.

The sample from which the maternal typing is performed can be any tissueincluding, but not limited to, nucleated blood cells, saliva and hairfollicles. It is preferred that the sample is obtained with as littlediscomfort to the mother as possible. In a particularly preferredembodiment, the sample comprises nucleated blood cells, such as T and Blymphocytes, macrophages etc, obtained from a blood sample taken fromthe mother.

The maternal typing should preferably be performed on a sample free fromfetal nucleic acids. Considering the quantity of fetal DNA typicallyfound in maternal plasma or serum samples it is preferred that suchmaternal plasma or serum samples are not used for typing the mother.Although the cellular fraction of a maternal blood sample has beenreported to contain fetal cells, it is known in the art that these fetalcells are not in a sufficiently high enough concentration to interferewith standard typing techniques. Thus, if the sample for maternal typingis contaminated with fetal nucleic acids the level of contamination willbe sufficiently low such that it does not interfere with the accuratetyping of the mother.

In a particularly preferred embodiment, a blood sample is taken from themother and the plasma and cellular fractions separated bycentrifugation, with the cellular fraction being utilized to type themother and the plasma fraction being used as the source of fetal nucleicacids.

Although it is preferable that typing the mother directly characterizesan allele, or protein (antigen) encoded thereby, the method may comprisetyping by analysing markers, such as single nucleotide polymorphisms orrepeat number polymorphisms, that are linked to a particular allele.Such linked polymorphic markers may be coding or non-coding.

Similarly, although it is preferred that the fetal nucleic acids fromthe sample comprising fetal and maternal nucleic acids are directlyanalysed for a specific allele(s), the method may comprise typing byanalysing markers, such as single nucleotide polymorphisms or repeatnumber polymorphisms, that are linked to a particular allele.

The allele may be any polymorphism existing within the population.Preferably, the polymorphism is within a gene. More preferably, thepolymorphism is within an exon of a gene which encodes a protein. Evenmore preferably, the polymorphism is within an exon of a gene whichencodes a protein such that the amino acid sequence of the encodedprotein varies between some members of the population.

In a particularly preferred embodiment, the allele is from an HLA locus.Thus, a preferred embodiment of the first aspect comprises a method ofidentifying an HLA allele of a fetal cell, said allele not being presentin maternal cells, the method comprising;

i) identifying HLA alleles which are not present in maternal cells,

ii) obtaining a sample from the mother comprising fetal and maternalnucleic acids, and

iii) screening nucleic acids from the sample for at least one HLA allelenot present in the maternal cells.

Furthermore, a preferred embodiment of the second aspect comprises amethod of identifying an HLA allele of a fetal cell, said allele notbeing present in maternal cells, the method comprising;

i) obtaining a sample from the mother comprising fetal and maternalnucleic acids,

ii) HLA typing at least one HLA locus from the sample obtained in stepi),

iii) comparing the HLA alleles identified in step with HLA alleles ofthe same locus possessed by the mother, and

iv) selecting an HLA allele typed in step which is not possessed by themother.

Any or all of the HLA genes may be analysed, including those of class I,class II or class III, using the method of the first or second aspect.Preferably, at least some of the class I or class II HLA alleles aretyped.

Screening the nucleic acids from the sample for at least one HLA allelespecific for a fetal cell can be performed using any technique known inthe art. In a preferred embodiment, the nucleic acid is subjected toamplification-based HLA typing procedures. In a particularly preferredembodiment, the nucleic acid is subjected to sequence specific primer(SSP) typing using a multitude of allele specific primer pairs.

As is known in the art, the HLA complex is highly polymorphic. Manypolymorphisms within, for example humans, have been characterized, andthe frequency within which a given allele exists in at least somespecific populations (e.g. geographic and/or race) has been determined.Upon characterizing a HLA allele of the mother at a given locus, knownpolymorphisms can readily be screened to determine relevant HLA alleleswhich occur in the population but are not possessed by the mother.

Preferably, screening nucleic acids from the sample for at least one HLAallele targets HLA alleles which are common in the population but absentin the mother. Naturally, this reduces the number of alleles which needto be analysed before a fetal DNA/cell marker is identified. Preferably,the targeted HLA allele is found within at least 5%, more preferably atleast 10% of the population.

The sample comprising fetal and maternal nucleic acids can be obtainedfrom any source known in the art. Examples include, but are not limitedto plasma, serum or urine. Preferably, the sample is derived from plasmaor serum. Preferably, the serum or plasma sample is subjected toaffinity chromatography to enrich nucleic acids from the sample beforethe method of the first or second aspect is performed.

The nucleic acid can be DNA or RNA. In the instance where the nucleicacid is RNA it is preferred that the RNA is reverse transcribed toproduce cDNA. However, it is particularly preferred that the nucleicacid is DNA.

Preferably the mother is a mammal. More preferably the mother is ahuman.

In another embodiment, the mother can be any organism which comprises aHLA-like complex. An example of a non-human HLA-like complex is themajor histocompatibility complex on chromosome 17 in the murine genome.

The fetal specific alleles identified using the methods of the inventioncan be used to quantify the amount of fetal nucleic acid in a samplethat comprises both fetal and maternal nucleic acids. As reported in theliterature, such quantification procedures are useful for the diagnosisof various diseases such as fetal trisomy 21.

Thus, in a third aspect the present invention provides a method ofquantifying fetal specific nucleic acids in a sample comprising fetaland maternal nucleic acids, the method comprising identifying an allelewhich is present in the DNA of a fetal cell but absent from maternal DNAusing a method according to the invention, and determining theconcentration of the allele in the sample.

The quantification of the fetal specific nucleic acid in the sample canbe performed using any technique known in the art. Preferably, theallele is quantified by exposing the sample to a nucleic amplificationprocedure which specifically amplifies the allele, and detecting theamplification product.

Preferably, the allele is an HLA allele.

Furthermore, in a fourth aspect the present invention provides a methodof screening for a disease associated with abnormal levels of fetal DNAin the mother, the method comprising identifying an allele which ispresent in the DNA of a fetal cell but absent from maternal DNA using amethod of the invention, and determining the concentration of the allelein a sample obtained from the mother which comprises fetal and maternalnucleic acids.

The abnormal levels of fetal DNA may be higher or lower than thosetypically found in healthy mothers carrying a fetus which is developingnormally. Examples of diseases which have been implicated in beingassociated with elevated levels of fetal DNA in the mother include, butare not limited to, fetal trisomy 21, fetal trisomy 13, preterm labour,preeclampsia, and idiopathic polyhydramnios.

Preferably, the allele is an HLA allele.

The isolation and/or detection of fetal cells from, for example maternalblood, requires a marker that is specific for the fetal cell. Ininstances where the allele identified by the first or second aspectencodes a protein, this protein can be considered as a fetal specificmarker.

Accordingly, in a fifth aspect the present invention provides a methodof isolating fetal cells from a sample, the method comprisingidentifying an allele, encoding an antigen, which is present in the DNAof a fetal cell but absent from maternal DNA using a method according tothe invention, binding to the fetal cell an affinity reagent whichrecognises the antigen, and selecting cells bound by the affinityreagent.

In a sixth aspect, the present invention provides a method of detectingfetal antigens in a sample, the method comprising identifying an allele,encoding an antigen, which is present in the DNA of a fetal cell butabsent from maternal DNA using a method according to the invention,exposing the sample to an affinity reagent which recognises the antigen,and detecting antigen-affinity reagent complexes.

In a seventh aspect, the present invention provides a method ofanalysing feto-maternal cell-trafficking and/or microchimerism, themethod comprising identifying an allele, encoding an antigen, which ispresent in the DNA of a fetal cell but absent from maternal DNA using amethod according to the invention, detecting a fetal cell in a sampleobtained from a mother by exposing the sample to an affinity reagentwhich recognises the antigen, and detecting antigen-affinity reagentcomplexes.

With regard to the seventh aspect, it is preferred that the disease isscleroderma. Furthermore, it is preferred that the mother has givenbirth to the child.

With regard to any one of the fifth, sixth or seventh aspects,preferably the sample is the cellular fraction of a blood sampleobtained from the mother.

Preferably, the antigen is a cell surface protein. More preferably, thecell surface protein is a HLA protein.

Preferably, the affinity reagent which recognises the antigen is anantibody. More preferably, the antibody is detectably labelled.Alternatively, antibody binding can be detected using a detectablylabelled secondary antibody. As is known in the art, the secondaryantibody binds the antibody directed against the antigen.

Examples of suitable detectable labels include, but are not limited to,those selected from the group consisting of a radioisotope, afluorescent compound, a colloidal metal, a chemiluminescent compound, abioluminescent compound, and an enzyme.

Also provided as an eighth aspect is a method of detecting fetalspecific nucleic acids in a sample, the method comprising identifying anallele which is present in the DNA of the fetal cell but absent frommaternal DNA using a method of the present invention, exposing thesample to an affinity reagent which recognises the allele, and detectingallele-affinity reagent complexes.

Preferably, the affinity reagent which recognises the allele is alabelled nucleic probe which selectively hybridizes to the allele.

The present inventor has also devised methods of quantifying fetalspecific nucleic acids in, for example, a maternal plasma or serumsample using subtractive hybridization based procedures. Accordingly, ina ninth aspect the present invention provides a method of quantifyingfetal specific nucleic acids in a sample comprising fetal and maternalnucleic acids, the method comprising using subtractive hybridization tocapture nucleic acids from the sample which are either maternal specificor which are shared between the mother and the fetus, and quantifyingthe remaining nucleic acids.

In one instance, the method relies on differential epigeneticmodifications between maternal and fetal DNA. Thus, in particularlypreferred embodiment the method of the ninth aspect comprises thefollowing

i) obtaining a first sample comprising maternal, but no or smallquantities of fetal, DNA,

ii) obtaining a second sample comprising fetal and maternal DNA,

iii) exposing the first and second sample to an agent that converts oneof, but not both, a) a nucleotide or b) the same nucleotide comprisingan epigenetic modification, in a manner such that upon synthesis of acomplementary sequence the complementary sequence is altered whencompared to synthesis in the absence of exposure to the agent,

iv) denaturing the DNA from the first sample from iii) and exposing theDNA to conditions which result in the synthesis of a complementarysequence, wherein the complementary sequence is DNA or RNA,

v) removing the DNA from iv) that was used as the template duringsynthesis,

vi) denaturing the DNA from the second sample from iii),

vii) combining the products of v) and vi) in the same vessel andexposing the products to conditions which promote nucleic acidhybridization,

viii) isolating and quantifying the DNA obtained from vii) which isderived from the second sample which has not hybridized to thesynthesized DNA or RNA produced in iv).

In this embodiment differences in epigenetic modifications are targetedto synthesize from maternally derived DNA a complementary sequence whichis sufficiently different to a complementary sequence synthesized fromthe corresponding locus of fetal DNA such that the two complementarysequences have differing ability to hybridize to a target moleculesynthesized using maternal DNA as a template. More specifically, where asuitable concentration of epigenetic differences exist between maternaland fetal DNA at a particular loci the method will result in the nucleicacid derived from the maternal loci being captured in vii), whilst thatderived from the fetal DNA will be isolated in step viii). Whilst atmany loci there will not be sufficient differences between epigeneticmodifications for the fetal derived molecules to be isolated in stepviii), there will be enough regions with such variations to enable stepviii) to result in the isolation of a population of nucleic acids whichcan be used as an indicator of total fetal DNA levels in the sample.

As the skilled addressee would be aware, numerous steps of thisembodiment can be rearranged without altering the working of theinvention. For example, ii) could be performed after v).

Preferably, the epigenetic modification is methylation of a cytosine.

Preferably, the agent is sodium bisulfite.

Preferably, v) further comprises attaching the synthesized DNA or RNAstrand to a solid support. Any suitable solid support may be used suchas, but not limited to, beads, plastics, silicon, a polymer matrix, or amembrane.

In a further preferred embodiment, the vessel has two open ends andcomprises therein the synthesized DNA or RNA derived from the firstsample attached to a solid support. In this embodiment, vii) comprisespassing the treated and denatured DNA from the second sample through thecolumn and collecting the eluate.

In yet another preferred embodiment, the synthesis results in theincorporation of a label molecule. The label can be used, for example,to attach the synthesized DNA or RNA to a solid support. In aparticularly preferred embodiment, at least some of the nucleotideprecursors used in the synthesis procedure are biotinylated, and thesynthesized products are attached to a solid support via the biotinlabel.

In a further preferred embodiment, upon denaturation of the DNA in thefirst sample, the synthesis is primed by short random oligonucleotideprimers (for example random hexamers) in the presence, of a suitablepolymerase such as, but not limited to, DNA polymerase I from E. coli.

In the instance where RNA is produced in the synthesis step, v) maycomprise exposing the reaction to DNase to remove the template DNA.

The hybridization and denaturing steps can be performed using techniquesknown in the art. Preferably, the hybridization is performed under highstringency conditions which promote only closely related sequences tohybridize.

Preferably, the amount of maternal DNA in the first sample is greaterthan the total DNA in the second sample (which will comprise both fetaland maternal DNA). More preferably, the amount of maternal DNA in thefirst sample is at least 20-fold higher, more preferably at least50-fold higher, and even more preferably at least 100-fold higher thanthe total DNA in the second sample.

The first sample can be obtained from any tissue of the mother whichcomprises no or small quantities of fetal DNA. As outlined above, somefetal cells may be present in a sample of nucleated blood cells obtainedfrom the mother. However, it is envisaged that such fetal cells will bepresent at insufficient concentration, namely as low as one fetal cellto a million maternal cells, to prevent the enrichment of fetal specificDNA. Preferably, the first sample is obtained from a tissue known not tocomprise fetal DNA or fetal cells such as saliva.

Preferably, the term comprises “small quantities of fetal DNA” meansthat maternal DNA is found at least at a 100 fold, more preferably atleast at a 1,000, and even more preferably at least at a 10,000 foldhigher concentration than any contaminating fetal DNA.

In a particularly preferred embodiment, the maternal DNA from the firstsample is cleaved into fragments.

Preferably, maternal DNA in the first sample is cleaved with arestriction endonuclease. Any restriction endonuclease, or a combinationthereof, may be used, however, it is preferred that the restrictionendonuclease cleaves the DNA to produce as many smaller fragments aspossible (but at least most fragments being 20 or more nucleotides inlength), such as a restriction endonuclease that cleaves at a 4 basepair recognition sequence. Preferably, any fragments smaller than about20 nucleotides in length are removed from the sample. This will minimizeany non-specific hybridization events.

Although DNA from the second sample will typically comprise DNAfragments as a result of numerous factors including non-sequencespecific endonucleases which act during cell death, the DNA from thesecond sample may also be cleaved with a suitable agent. In a preferredembodiment, the DNA in the second sample is cleaved with the samerestriction enzyme(s) as the DNA in the first sample.

The DNA can be quantified using any technique known in the art. In oneembodiment, the DNA is quantified through the use of an agent whichbinds the DNA. Preferably, the agent is picogreen. In some cases theagent may preferentially bind dsDNA. Thus, in these instances to ensurethat the DNA quantified is dsDNA, the isolated DNA from viii) may beused as a template to synthesize a complementary strand thereto and theresulting dsDNA products quantified.

In another instance, a particularly preferred embodiment of the methodof the ninth aspect comprises the following

i) obtaining a first sample comprising maternal, but no or smallquantifies of fetal, DNA,

ii) obtaining a second sample comprising fetal and maternal DNA,

iii) denaturing the DNA obtained from i) and

iv) exposing the denatured DNA of the first and second samples in thesame vessel to conditions which promote nucleic acid hybridization, and

v) isolating and quantifying the DNA obtained from iv) which is derivedfrom the second sample which has not hybridized to DNA from the firstsample.

As the skilled addressee would be aware, numerous steps of thisembodiment can be rearranged without altering the working of theinvention. For example, i) could be performed after ii).

The DNA in the first sample of this embodiment is not only used toremove, at least partially, maternal DNA from the second sample but willalso remove, at least partially, fetal DNA which has been inherited fromthe mother, as well as remove, at least partially, fetal DNA which hasbeen inherited from the father which is the same or shares a high degreeof sequence identity with DNA of the mother.

In a tenth aspect, the present invention provides a method of screeningfor a disease associated with abnormal levels of fetal DNA in themother, the method comprising quantifying fetal DNA in a sample using amethod of the ninth aspect.

Preferably, the disease is selected from, but not limited to, fetaltrisomy 21, fetal trisomy 13, preterm labour, preeclampsia, andidiopathic polyhydramnios.

The present inventor has also devised a method for at least partiallypurifying fetal specific nucleic acids from a sample comprising fetaland maternal nucleic acids. This method relies, in part, on removingmaternal DNA from the sample through nucleic hybridization procedures.Thus, in an eleventh aspect the present invention provides a method ofenriching from a sample a DNA sequence which is present in fetal DNA butabsent in maternal DNA, the method comprising;

i) obtaining a first sample comprising maternal, but no or smallquantities of fetal, DNA,

ii) obtaining a second sample comprising fetal and maternal DNA,

iii) denaturing the DNA obtained from i) and ii),

iv) exposing the denatured DNA of the first and second samples in thesame vessel to conditions which promote nucleic acid hybridization, and

v) collecting the non-hybridized DNA which is enriched in fetal specificDNA when compared to the second sample.

As used herein, the term “enriching” refers to the relativeconcentration (when compared to maternal DNA) of fetal specific DNAbeing increased compared to the relative concentration (when compared tomaternal DNA) of fetal specific DNA in the second sample.

The enrichment aspect of the invention can be used in the methods of theinvention related to the identification of fetal specific HLA alleles.However, the enrichment aspect may also be used to identify non-HLAmarkers of the fetal cells. Thus, in a twelfth aspect the presentinvention provides a method of identifying a DNA sequence which ispresent in fetal DNA but absent in maternal DNA, the method comprising;

i) obtaining a first sample comprising maternal, but no or smallquantities of fetal, DNA,

ii) obtaining a second sample comprising fetal and maternal DNA,

iii) denaturing the DNA obtained from i) and ii),

iv) mixing and exposing the denatured DNA of the first and secondsamples in the same vessel to conditions which promote nucleic acidhybridization,

v) collecting the non-hybridized DNA which is enriched in fetal specificDNA when compared to the second sample, and

vi) screening DNA from v) for non-maternal DNA sequences.

As the skilled addressee would be aware, numerous steps of this aspectcan be rearranged without altering the working of the invention. Forexample, i) could be performed after ii).

The screening for non-maternal DNA as defined in step vi) can beperformed by any technique known in the art. In one embodiment, the DNAfrom step v) is exposed to DNA amplification using primers directed to alocus of interest In an alternate embodiment, the DNA from step v) isplaced on a solid support, such as used in microarray technology, andscreened with allele specific probes for a locus of interest.Preferably, the locus of interest is known to be polymorphic betweenindividuals, such as loci of the HLA complex.

The method of the twelfth aspect can also be applied to RNA in a fetalcell which is differentially expressed when compared to a maternal cell.Thus, in a thirteenth aspect, the present invention provides a method ofidentifying an RNA sequence which is present in a fetal cell but absentin a maternal cell, the method comprising;

i) obtaining a first sample comprising maternal, but no or smallquantities of fetal, RNA,

ii) obtaining a second sample comprising fetal and maternal RNA,

iii) reverse transcribing the RNA in the first and second samples toproduce cDNA,

iii) denaturing the cDNA obtained from iii),

iv) mixing and exposing the denatured cDNA of the first and secondsamples in the same vessel to conditions which promote nucleic acidhybridization,

v) collecting the non-hybridized cDNA which is enriched in fetalspecific cDNA when compared to the second sample, and

vi) screening cDNA from v) for sequences derived from fetal specificRNA.

Preferably, the term comprises “small quantities of fetal RNA” meansthat maternal RNA is found at least at a 100 fold, more preferably atleast at a 1,000, and even more preferably at least at a 10,000 foldhigher concentration than any contaminating fetal RNA.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1: HLA-A typing of maternal-cellular and plasma-derived DNA. Imagesshow the results of reactions targeted to eight single types includingA01, A02, A03, A11, A24, A26, A29 and A32. For each sample the left handimage indicates the types identified in maternal-cellular-derived DNA,while the right hand image identifies types found in plasma-derived DNA.HLA-A products are between 160 and 240 bp. The lane marked ‘M’ is a 100bp DNA size ladder.

FIG. 2: HLA-A typing of maternal-cellular; paternal-cellular andmaternal plasma-derived DNA. Panels A & B show the results of reactionstargeted to eight single types including A01, A02, A03, A11, A24, A26,A29 and A32. Panel A shows typing of maternal DNA (sample 502). Panel Bshows typing of paternal DNA (sample JK). Panel C shows reactionstargeted to the paternal types (A29/A32) using plasma-derived DNA,maternal DNA and paternal DNA. HLA-A products are between 160 and 240bp. The lane marked ‘M’ is a size ladder.

DETAILED DESCRIPTION OF THE INVENTION General Techniques

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, immunology, nucleicacid chemistry, hybridization techniques and biochemistry).

Unless otherwise indicated, the recombinant DNA and immunologicaltechniques utilized in the present invention are standard procedures,well known to those skilled in the art. Such techniques are describedand explained throughout the literature in sources such as, J. Perbal, APractical Guide to Molecular Cloning, John Wiley and Sons (1984), J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present), and are incorporated herein by reference.

As used herein, the term “high stringency conditions” for hybridizationrefers to, but is not limited to, conditions such as 50% formamide,6×SSC at 42° C. (or other similar hybridization solution), followed bywashing conditions of about 60-68° C., in 0.2×SSC, 0.1% SDS (seeSambrook et al. (1989) and Ausubel et al. (1988) supra).

As used herein, the term “fetal specific” or variations thereof merelyrefers to an allele or protein which is possessed by a fetus of apregnant female but said allele or protein is does form part of (notencoded by in the case of proteins) the mothers genome.

Genotyping/Typing

The locus (loci) analysed using the methods of the first or secondaspect can be any locus (loci) known to be, or determined to be,polymorphic between different members of a population. Examples of suchpolymorphic loci are well known in the art. In some cases thepolymorphic loci are linked to a disease state, however, in manyinstances the polymorphic loci are not linked to a disease state. In oneembodiment, the polymorphism is a single nucleotide polymorphism (SNP).Examples of SNPs that could be alleles targeted by the methods of theinvention to identify a fetal specific nucleic acid in a pregnant femaleare provided in many sources including databases such as available athttp://www.ncbi.nlm.nih.gov/SNP/index.html.

Alleles at a given locus can be detected using any method known in theart, including PCR, nucleic acid sequencing and antibody detectionsystems, generally described below in relation to typing HLA alleles.

HLA Typing

HLA typing can be performed using any technique known in the art. Ageneral overview of such procedures is provided in Parham et al. (1999),Gerlach (2002), as well as Hui and Bidwell (1993). These techniquesinclude serological and molecular typing, or a combination thereof,procedures. The mother can be typed using any procedure, althoughmolecular typing procedures are generally replacing the use ofserological techniques. In contrast, as the fetal material to beanalysed is nucleic acid the typing procedures in this instance will beusing nucleic acid detection based techniques.

There are many commercial suppliers of “kits” and/or reagents for HLAtyping. These include, but are not limited to, Dynal Biotech (Oslo,Norway), Pel-Freez Clinical Systems, LLC (Wisconsin, USA), Biotest(Frankfurt, Germany), Forensic Analytical (California, USA), andInnogenetics (Gent, Belgium). Such kits and/or reagents can be used inthe methods of the invention.

An example of a standard procedure used for serological HLA typing isthe lymphocyte microcytoxicity assay (Hui and Bidwell, 1993) which isalso known the art as complemement-dependent cytotoxicity assay. In suchan assay anti-HLA serum, or monoclonal antibody, is mixed with livelymphocytes obtained from the mother. Specific Ig binds to thepolymorphic protein moiety of the HLA molecule expressed on the cellsurface. Exogenous complement is added to the well which will result inlysis of cells to which antibody has been bound. Cell death isdetermined by, for example, ethidium bromide vital stain exclusion.Specific binding is detected by, for example, vital dye stain. Vital dyeexclusion techniques, which are commonly used, often require millions ofviable lymphocytes that express HLA antigens which makes them difficultto perform in small children or patients with low white cell counts.Accordingly, the small number of fetal cells which may be in a sampleobtained from the maternal blood will not influence the correct HLAtyping of the mother using this procedure.

Many methods based on nucleic acid amplification are currently used forthe detection of HLA alleles (Mike et al., 1997). Such moleculartechniques can be catergorised into at least four types: i) methods thatgenerate a product containing internally located polymorphisms that areidentified by a second technique (for example, sequence specificoligonucleotide probes (SSOP)); ii) methods in which the polymorphism isidentified directly as part of the PCR process although there arepost-amplification steps (for example the use of sequence specificprimers); iii) methods which analyse different mutations generatingspecific conformation changes (heteroduplexes), and iv) Methods thatrely on DNA sequencing. The choice of method in a laboratory will dependon a number of factors including, but not limited to, clinical urgency,resolution, sample numbers, budget, equipment availability, and staffskills.

Amplification of DNA followed by probing with sequence-specificoligonucleotide probes (SSOP) can be performed using several relatedprocedures. The principle of this approach is that different alleles ata locus can be detected by probes recognizing the allelic differencesand used to detect the complementary region of an amplified target. Itis customary to have one of the DNA strands in the reaction immobilized.However, procedures where both immobilization of the target and theprobe motifs have been developed. If the amplified product isimmobilized, then the probe is labeled and applied. This is atraditional dot blot, named such because the target is spotted as dotson a filter or membrane. The material can also be applied as a line, andthe process is then referred to as a slot blot. If the probe isimmobilized and the amplified target is labeled, the assay is thenreferred to as a reverse dot blot or a “blot dot.” These methods arewell suited to high-throughput needs, as many samples can be spotted andprobed in a single reaction in the dot-blot scenario, and a sample canbe reacted with many probes in one reaction in a blot-dot paradigm. Touse many probes at once, the temperature of melting for the probes needsto be optimized.

Amplification of an allele at a locus using allele-specific primers isalso used extensively. This method is often referred to assequence-specific primer (SSP) or amplification refractory mutationsystem (ARMS) typing. While SSP and ARMS have a slightly differentcontext, the principle is very much the same. In these systems, theamplification process is performed using one primer that is conservedacross many alleles and a second primer specific for a single nucleotidepolymorphism. The 3′ end of a primer must be firmly bound to the targetfor the polymerase to extend the strand. The detection employed in SSPor ARMS methods is usually an agarose gel fractionation of products.Most kits available have multiple primer sets in separate reactions totype for many alleles at a locus or loci. Temperature of meltingoptimization of the primers is necessary to allow thermal cycling of allreactions in a single instrument. Testing using SSP and ARMS can yieldlow-, medium-, or high-resolution results, depending on the primer setsused.

Given the proper physical environment, DNA will form secondarystructures. The placement of the “loops” and “hairpins” that form aresequence dependent. If 2 alleles have different sequences, the types andplacement of secondary structures should differ. This is the basis ofdouble-stranded sequence conformation polymorphism testing. The conceptis that alleles allowed to form secondary structures can bediscriminated among themselves by the migration of these secondarystructures in a gel matrix environment. Resolution can be enhanced byutilizing a reference strand in the duplex formations. This serves as aninternal reference across all allele combinations, because only thoseduplexes with the reference strand are visualized.

The advent of capillary sequencers has made HLA typing by sequencingmore economical and gives a quicker and larger throughput than previoussequencing technologies.

In the future many of the techniques now evolving to ascertain singlenucleotide polymorphisms (SNP) will be applied in the HLA field e.g.wave technology, mass spectrometry, and microarrays. Furthermore, manymethods will become automated.

As outlined above, it is preferred that screening the nucleic acids fromthe sample for at least one HLA allele targets HLA alleles which arecommon in the population but absent in the mother. Naturally, thisreduces the number of alleles which need to be analysed before a fetalDNA/cell marker is identified. Preferably, the targeted HLA allele isfound within at least 5%, more preferably 10% of the population.Information regarding known HLA alleles, as well as their frequency 5within the population, can be determined using standard typingprocedures, the results of which have been widely reported (see, forexample, Middleton et al. (2000); Williams et al. (2002); Marsh et al.2002; www.ebi.ac.uk/imgt/hla—Robinson et al., 2003;www.allelefrequencies.net—Middleton et al., 2002).

Fetal DNA Isolation and Analysis Thereof

Fetal DNA can be obtained from the sample using any technique known inthe art. Preferably, the sample is maternal plasma or serum obtainedduring the first trimester of pregnancy. A review of fetal DNA inmaternal plasma and serum is provided by Pertl and Bianchi (2001), aswell as Lo (2000).

As outlined in WO 98/39474, maternal plasma can be obtained from wholematernal blood by separating the cellular fraction throughcentrifugation at 3000 g. Following centrifugation the plasma layer isremoved and placed into a polypropylene tube containing ananti-coagulant such as EDTA. Although DNA can be extracted from theplasma sample using standard techniques involving, for example ethanolprecipitation or affinity column purification (such as those produced byQiagen, California, USA), molecular analysis such as nucleic acidamplification can be performed essentially directly on the plasma orserum sample.

Molecular techniques for the analysis of fetal nucleic acids are theessentially same as the molecular techniques described above in the HLAtyping section. As outlined in Hahn et al. (2001), the TaqMan system(Applied Biosystems, California, USA) has shown to provide robust andreproducible results.

Nucleic Acid Quantification

Nucleic acids identified/isolated using methods of the invention can bequantitated using any technique known in the art.

In one embodiment, upon the identification of a fetal specific allelewhich is not present in the mother a quantitative polymerase chainreaction (QPCR) can be performed, for example on a maternal plasma orserum sample, to determine the relative quantity of the allele in thesample. This in turn can be used as a marker for diseases such as fetaltrisomy 21.

QPCR is a method for quantifying a nucleic acid molecule based ondetection of a fluorescent signal produced during PCR amplification(Gibson et al., 1996). Amplification is carried out on machines such asthe PRISM 7700 detection system (ABI) which consists of a 96-wellthermal cycler connected to a laser and charge-coupled device (CCD)optics system. To perform QPCR, a PCR reaction is carried out in thepresence of a doubly labeled probe. The probe, which is designed toanneal between the standard forward and reverse PCR primers, is labeledat the 5′ end by a fluorogenic reporter dye such as 6-carboxyfluorescein(6-FAM) and at the 3′ end by a quencher molecule such as6-carboxy-tetramethyl-rhodamine (TAMRA). As long as the probe is intact,the 3′ quencher extinguishes fluorescence by the 5′ reporter. However,during each primer extension cycle, the annealed probe is degraded as aresult of the intrinsic 5′ to 3′ nuclease activity of Taq polymerase.This degradation separates the reporter from the quencher, andfluorescence is detected every few seconds by the CCD. The higher, thestarting copy number of the nucleic acid, the sooner an increase influorescence is observed. A cycle threshold (CT) value, representing thecycle number at which the PCR product crosses a fixed threshold ofdetection is determined by the instrument software. The CT is inverselyproportional to the copy number of the template and can therefore beused to calculate either the relative or absolute initial concentrationof the nucleic acid molecule in the sample. The relative concentrationof two different molecules can be calculated by determining theirrespective CT values (comparative CT method). Alternatively, theabsolute concentration of the nucleic acid molecule can be calculated byconstructing a standard curve using a housekeeping molecule of knownconcentration. The process of calculating CT values, preparing astandard curve, and determining starting copy number can be performedusing SEQUENCE DETECTOR 1.7 software (ABI).

Examples of QPCR procedures which are useful for the present inventioninclude, but are not limited to, those generally described by Honda etal. (2002), Lo et al. (1999), Lee et al. (2002), Wataganara et al.(2003), and Zhong et al. (2000).

Nucleic acids may also be quantified by using agents which bind thereto.Examples of such agents include, but are not limited to, ethidiumbromide, Hoechst 33258 and picogreen. In a preferred embodiment, theagent is picogreen (Ahn et al., 1996).

In another embodiment the nucleic acids are quantified using an antibodywhich is specific therefor but which binds independent of the actualnucleotide sequence. An example of an antibody which is specific for,and can be used to quantify, ssDNA is described by Batova et al. (1993).

In a further embodiment, particularly with regard to DNA obtained fromthe method of the ninth aspect, the DNA can be quantified by ligatingadapter fragments to the end of the DNA and using primers directedagainst the adapters to amplify the ligated DNA in a QPCR procedure(see, for example, Zou et al., 2003).

Fetal Cell Isolation

At least some fetal cell types such as platelets, trophoplasts,erythrocytes and leucocytes have been shown to cross the placenta andcirculate in maternal blood (Douglas et al., 1959; Schroder, 1975). Suchfetal cells can provide fetal DNA for prenatal genetic testing. Fetalcell isolation requires a unique marker for fetal cells, which arepresent in maternal blood at a frequency of less than 1 fetal cell in1,000,000 maternal cells.

The identification of a fetal cell marker by the methods of theinvention can be used to assist in fetal cell isolation. Methods areavailable for selecting cells using markers which are either inside thecell or on the cell surface. In a preferred embodiment, the fetal cellmarker is localised on the cell surface. In particular, as with numerousaspects of the present invention, the fetal cell marker is an HLAantigen.

Preferably, the sample comprising fetal cells is obtained from apregnant woman in her first trimester of pregnancy. In one embodimentthe sample can be a blood sample which is prevented from clotting suchas a sample containing heparin or, preferably, ACD solution. The sampleis preferably stored at 0 to 4° C. until use to minimize the number ofdead cells, cell debris and cell clumps. The number of fetal cells inthe sample varies depending on factors including the age of the fetus.Typically, from 7 to 20 ml of maternal blood provides sufficient fetalcells upon separation from maternal cells. Preferably, 30 ml or moreblood is drawn to ensure sufficient cells without the need to draw anadditional sample.

In another embodiment, the fetal cells are obtained from the cervicalmucous of the mother as, for example, generally described in WO03/020986.

Before being selected using a method of the invention, the fetal cellsmay at least be partially purified from a sample obtained from themother by a procedure such as that described in WO 03/102595.

A fetal cell specific affinity reagent is used to isolate fetal cellsaway from maternal cells present in the central maternal blood sample.Isolation may be accomplished by a variety of techniques well known inthe art, including cell sorting, especially fluorescence-activated cellsorting (FACS), by using an affinity reagent bound to a substrate (e.g.,a plastic surface, as in panning), or by using an affinity reagent boundto a solid phase particle which can be isolated on the basis of theproperties of the beads (e.g., colored latex beads or magneticparticles). As will be apparent to one of skill in the art, the fetalcell affinity reagent may be bound directly or indirectly (e.g., via asecondary antibody) to the dye, substrate, or particle. In aparticularly preferred embodiment, the fetal cell affinity reagent is anantibody that binds a HLA antigen of the fetal cell, wherein thematernal cells do not express the antigen.

For isolation of fetal cells by cell sorting, the affinity reagent islabeled directly or indirectly with substance which can be detected by acell sorter, preferably a dye. Preferably, the dye is a fluorescent dye.A large number of different dyes are known in the art, includingfluorescein, rhodamine, Texas red, phycoerythrin, and the like. Anydetectable substance which has the appropriate characteristics for thecell sorter may be used (e.g., in the case of a fluorescent dye, a dyewhich can be excited by the sorter's light source, and an emissionspectra which can be detected by the cell sorter's detectors).

For isolation of fetal cells using solid-phase particles, any particlewith the desired properties may be utilized. For example, largeparticles (e.g., greater than about 90-100 μm in diameter) may be usedto facilitate sedimentation. Preferably, the particles are “magneticparticles” (i.e., particles which can be collected using a magneticfield). Magnetic particles are now commonly available from a variety ofmanufacturers including Dynal Biotech (Oslo, Norway). An example ofmagnetic cell sorting (MACS) is provided by Al-Mufti et al. (1999).

When a dye or a solid phase particle is used in conjunction with theaffinity reagent to isolate fetal cells from the central maternal bloodsample, the dye or solid phase particle may be directly or indirectlylinked to the affinity reagent. Whether the affinity reagent is directlyor indirectly linked is left to the discretion of the practitioner.Directly labeled affinity reagents are produced by linking the dye orsolid phase particle to the affinity reagent by, for example, covalentlinkage of a dye or by adsorption to a solid phase particle. Affinityreagents may be indirectly labeled using a variety of methods known inthe art, such as using a “secondary antibody” (a directly labeledantibody which binds specifically to the affinity reagent), or byexploiting a binding pair such as biotin and streptavidin (e.g., byderivatizing the affinity reagent with biotin, and using directlylabeled streptavidin eptavidin to bind label to the affinity reagent).

For isolation of fetal cells using an affinity reagent bound to asubstrate, the affinity reagent is preferably adsorbed or bound directlyto the substrate. Preferably, the substrate is the surface of a plasticplate or flask, and the affinity reagent is directly adsorbed to thesurface. Adsorption is easily accomplished for most affinity reagents,and when the affinity reagent is an antibody, adsorption is accomplishedby simply incubating a solution containing the antibody on thesubstrate. Alternately, a modified substrate may be used, such as asubstrate modified with avidin or streptavidin, and an affinity reagentmodified with biotin, or an amine-derivatized substrate activated with abifunctional crosslinking agent. Preferably, the affinity reagent isadsorbed to the substrate by incubating a solution containing theaffinity reagent on the substrate.

Fetal cell isolation may be aided by the depletion of maternal cellsprior to fetal cell sorting. In this case the mononuclear cell layer canbe initially isolated from the blood of pregnant women bycentrifugation. The resulting cell suspension consists predominantly ofmaternal cells; in order to enrich the eventual proportion of fetalcells present, the maternal cells are selectively removed by incubatingthe cells with antibodies attached to a solid support. Such supportsinclude magnetic beads, plastic flasks, plastic dishes and columns. Theantibodies bind antigens present on the cell surface of matureleukocytes. Thus, a non-trivial number of maternal leukocytes areeliminated by virtue of being bound to the solid support. The totalnumber of cells remaining in the cell suspension is smaller, but theproportion of fetal cells present is larger.

As outlined above, preferably the affinity reagent is an antibody orfragment or derivative thereof.

Monoclonal antibodies which will bind to HLA antigens are already knownbut in any case, with today's techniques in relation to monoclonalantibody technology, antibodies can be prepared to most antigens. Theantigen-binding portion may be a part of an antibody (for example a Fabfragment) or a synthetic antibody fragment (for example a single chainFv fragment [ScFv]). Suitable monoclonal antibodies to selected antigensmay be prepared by known techniques, for example those disclosed in“Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press,1988) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982).

Polyclonal antibodies are useful in the methods of the invention.Monospecific polyclonal antibodies are preferred. Suitable polyclonalantibodies can be prepared using methods well known in the art.

Fragments of antibodies, such as Fab and Fab₂ fragments may also be usedas can genetically engineered antibodies and antibody fragments.

The variable heavy (V_(H)) and variable light (V_(L)) domains of theantibody are involved in antigen recognition. Variable domains of rodentorigin may be fused (“humanized”) to constant domains of human originsuch that the resultant antibody retains the antigenic specificity ofthe rodent parented antibody.

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules; Fvmolecules; single-chain Fv (ScFv) molecules where the V_(H) and V_(L)partner domains are linked via a flexible oligopeptide, and singledomain antibodies (dAbs) comprising isolated V domains.

Fab, Fv, ScFv and dAb antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent, having only one antigen combining site.

After isolation using the affinity reagent, the isolated fetal cells maybe used directly for prenatal genetic testing, or they may be culturedto expand cells numbers and to facilitate karyotypic analysis.

Uses

Numerous conditions have been linked to abnormal amounts of fetal DNA inmaternal blood, such conditions include, but are not limited to, fetaltrisomy 21 (Down syndrome) (Lee et al., 2002), fetal trisomy 13(Wataganara et al., 2003), preterm labour (Leung et al., 1998),preeclampsia (Lo et al., 1999), and idiopathic polyhydramnios (Zhong etal., 2000) (for a review see Pertl and Bianchi, 2001).Gender-independent fetal DNA markers identified by the methods of thepresent invention can be used to quantify fetal DNA in, for example, theplasma/serum of a mother to detect conditions/diseases, such as thoseoutlined above, associated with abnormal levels of fetal DNA.

In addition, the fetal cell markers identified by the methods of thepresent invention can be used to isolate fetal cells. For example,antibodies which bind HLA antigens which are fetal specific (i.e.determined by the methods of the invention to be specific for a givenfetus when compared to the maternal HLA type) can be used in, forexample, flow cytometry procedures to isolate fetal cells. Because suchisolated fetal cells comprise the same genetic DNA make up of thesomatic cells of the fetus, these isolated fetal cells can be analysedfor abnormalities using techniques known in the art. Such analysis canbe performed on any cellular material that enables defects to bedetected. Preferably, this material is nuclear DNA, however, at least issome instances it may be informative to analyse RNA or protein from theisolated fetal cells. Furthermore, the DNA may encode a gene, or mayencode a functional RNA which is not translated, or the DNA analysed mayeven be an informative non-transcribed marker.

In one preferred embodiment, chromosomal abnormalities are detected. By“chromosomal abnormality” we include any gross abnormality in achromosome or the number of chromosomes. For example, this includesdetecting trisomy in chromosome 21 which is indicative of Down'ssyndrome, trisomy 18, trisomy 13, sex chromosomal abnormalities such asKlinefelter syndrome (47, XXY), XYY or Turner's syndrome, chromosometranslocations and deletions, a small proportion of Down's syndromepatients have translocation and chromosomal deletion syndromes includePradar-Willi syndrome and Angelman syndrome, both of which involvedeletions of part of chromosome 15, and the detection of mutations (suchas deletions, insertions, transitions, transversions and othermutations) in individual genes. Other types of chromosomal problems alsoexist such as Fragile X syndrome which can be detected by DNA analysis.

Other genetic disorders which can be detected by DNA analysis are knownsuch as 21-hydroxylase deficiency or holocarboxylase synthetasedeficiency, aspartylglucosaminuria, metachromatic leukodystrophyWilson's disease, steroid sulfatase deficiency, X-linkedadrenoleukodystrophy, phosphorylase kinase deficiency (Type VI glycogenstorage disease) and debranching enzyme deficiency (Type III glycogenstorage disease). These and other genetic diseases are mentioned in TheMetabolic and Molecular Basis of Inherited Disease, 7th Edition, VolumesI, II and III, Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D.(eds), McGraw Hill, 1995. Clearly, any genetic disease where the genehas been cloned and mutations detected can be analysed.

Genetic assay methods include the standard techniques of karyotyping,analysis of methylation patterns, restriction fragment lengthpolymorphism assays and PCR-based assays, as well as other methodsdescribed below.

Chromsomal abnormalities can be detected by karyotyping which is wellknown in the art. Karyotyping analysis is generally performed on cellswhich have been arrested during mitosis by the addition of a mitoticspindle inhibitor such as colchicine. Preferably, a Giemsa-stainedchromosome spread is prepared, allowing analysis of chromosome number aswell as detection of chromosomal translocations.

The genetic assays may involve any suitable method for identifyingmutations or polymorphisms, such as: sequencing of the DNA at one ormore of the relevant positions; differential hybridisation of anoligonucleotide probe designed to hybridise at the relevant positions ofeither the wild-type or mutant sequence; denaturing gel electrophoresisfollowing digestion with an appropriate restriction enzyme, preferablyfollowing amplification of the relevant DNA regions; S1 nucleasesequence analysis; non-denaturing gel electrophoresis, preferablyfollowing amplification of the relevant DNA regions; conventional RFLP(restriction fragment length polymorphism) assays; selective DNAamplification using oligonucleotides which are matched for the wild-typesequence and unmatched for the mutant sequence or vice versa; or theselective introduction of a restriction site using a PCR (or similar)primer matched for the wild-type or mutant genotype, followed by arestriction digest. The assay may be indirect, ie capable of detecting amutation at another position or gene which is known to be linked to oneor more of the mutant positions. The probes and primers may be fragmentsof DNA isolated from nature or may be synthetic.

A non-denaturing gel may be used to detect differing lengths offragments resulting from digestion with an appropriate restrictionenzyme. The DNA is usually amplified before digestion, for example usingthe polymerase chain reaction (PCR) method and modifications thereof.

Amplification of DNA may be achieved by the established PCR methods orby developments thereof or alternatives such as the ligase chainreaction, QB replicase and nucleic acid sequence-based amplification.

An “appropriate restriction enzyme” is one which will recognise and cutthe wild-type sequence and not the mutated sequence or vice versa. Thesequence which is recognised and cut by the restriction enzyme (or not,as the case may be) can be present as a consequence of the mutation orit can be introduced into the normal or mutant allele using mismatchedoligonucleotides in the PCR reaction. It is convenient if the enzymecuts DNA only infrequently, in other words if it recognises a sequencewhich occurs only rarely.

In another method, a pair of PCR primers are used which hybridise toeither the wild-type genotype or the mutant genotype but not both.Whether amplified DNA is produced will then indicate the wild-type ormutant genotype (and hence phenotype).

A preferable method employs similar PCR primers but, as well ashybridsing to only one of the wild-type or mutant sequences, theyintroduce a restriction site which is not otherwise there in either thewild-type or mutant sequences.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme sites appended to their 5′ ends.Thus, all nucleotides of the primers are derived from the gene sequenceof interest or sequences adjacent to that gene except the fewnucleotides necessary to form a restriction enzyme site. Such enzymesand sites are well known in the art. The primers themselves can besynthesized using techniques which are well known in the art. Generally,the primers can be made using synthesizing machines which arecommercially available.

PCR techniques that utilize fluorescent dyes may also be used to detectgenetic defects in DNA from fetal cells isolated by the methods of theinvention. These include, but are not limited to, the following fivetechniques.

i) Fluorescent dyes can be used to detect specific PCR amplified doublestranded DNA product (e.g. ethidium bromide, or SYBR Green I).

ii) The 5′ nuclease (TaqMan) assay can be used which utilizes aspecially constructed primer whose fluorescence is quenched until it isreleased by the nuclease activity of the Taq DNA polymerase duringextension of the PCR product.

iii) Assays based on Molecular Beacon technology can be used which relyon a specially constructed oligonucleotide that when self-hybridizedquenches fluorescence (fluorescent dye and quencher molecule areadjacent). Upon hybridization to a specific amplified PCR product,fluorescence is increased due to separation of the quencher from thefluorescent molecule.

iv) Assays based on Amplifluor (Intergen) technology can be used whichutilize specially prepared primers, where again fluorescence is quencheddue to self-hybridization. In this case, fluorescence is released duringPCR amplification by extension through the primer sequence, whichresults in the separation of fluorescent and quencher molecules.

v) Assays that rely on an increase in fluorescence resonance energytransfer can be used which utilize two specially designed adjacentprimers, which have different fluorochromes on their ends. When theseprimers anneal to a specific PCR amplified product, the twofluorochromes are brought together. The excitation of one fluorochromeresults in an increase in fluorescence of the other fluorochrome.

Fetal cells have been found to persist for decades post-partum, and arecapable of further differentiation and migration into maternal organs.As a result, the mother can be considered as a chimera (for a review seeBianchi et al. 2000). This phenomenon has been associated with variousdiseases, particularly autoimmune type diseases. Such diseasesassociated with feto-maternal cell-trafficking and/or microchimerism(for example scleroderma) can also be analysed using the methods of thepresent. More specifically, the fetal cell specific markers identifiedby the methods of the invention can be used to locate, quantify, captureand/or characterize fetal cells in the mother following birth or loss ofthe fetus.

EXAMPLES Example 1 Materials and Methods

Isolation of DNA from PlasmaPreparation of Cell-Free Plasma from Whole Blood

-   -   1. Centrifuge whole blood at 1000×g for 10 minutes    -   2. Allow cells to settle for a further 10 minutes    -   3. Remove plasma phase with a pipette    -   4. Re-centrifuge the plasma phase at 1600×g for 10 minutes    -   5. Aliquot plasma into 1.5 ml Eppendorf tubes    -   6. Centrifuge at 16000×g for 5 minutes    -   7. Transfer cleared plasma to a clean tube for DNA extraction or        storage.        DNA extraction

Genomic DNA was extracted from 200 μl of plasma using the Qiagen QIAmpDNA Blood minikit, following the Blood and Body Fluids protocol with thefollowing modifications. The DNA solution was passed through a singlecolumn four (4) consecutive times before the wash steps. DNA was elutedfrom the column using 65 μl of warm (56° C.) 1× PCR buffer (Invitrogen).The eluate was re-loaded onto the column for a second elution. Multipleextractions (200 μl per column) from the same plasma sample were pooledfor use in the HLA-A PCR reactions.

A detailed protocol follows:

-   -   1. Add 200 μl Plasma to a 1.5 ml tube    -   2. Add 20 μl of Qiagen Protease (or proteinase K)    -   3. Add 200 μl of Qiagen buffer AL    -   4. Incubate at 56° C. for 10-30 minutes    -   5. Add 200 μl 100% Ethanol and mix well    -   6. Apply to a Qiagen QIAmp DNA Blood Minikit column and spin at        3000 rpm for 1 minute    -   7. Re-apply the eluate to the same column and spin at 3000 rpm        for 1 minute    -   8. Repeat step 7. twice more (i.e., a total of four passes        through the column)    -   9. Place column in a fresh collection tube and add 500 μl of        Qiagen buffer AW1 then spin at full speed for 1 minute    -   10. Place column in a fresh collection tube and add 500 μl of        Qiagen buffer AW2 then spin at full speed for 1 minute    -   11. Empty collection tube and re-spin column at full speed for 2        minutes to dry    -   12. Add 65 μl of Qiagen elution buffer (pre-warmed to 56° C.),        allow to sit for at least 1 minute and spin at full speed for 1        minute    -   13. Eluants from multiple 200 μl aliquots of a single plasma        sample, processed with separate columns, are added back onto        each column for a final elution, and then pooled.    -   14. Store DNA at −20° C.    -   15. 20 μl aliquots of the pooled DNA solution are to be entered        into the targeted PCR reactions.        Extraction of DNA from Whole Blood

Genomic cellular DNA is extracted from 200 μl of whole blood using theQiagen QIAamp DNA Blood minikit according to the manufacturersinstructions and including two 100 μl elution's in warm. (56° C.)elution buffer. Detailed protocol is as follows:

-   -   1. Add 200 μl of whole blood to a 1.5 ml eppendorf tube    -   2. Add 20 μl of Qiagen Protease (or proteinase K)    -   3. Add 200 μl of Qiagen buffer AL    -   4. Incubate at 56° C. for 10-30 minutes    -   5. Add 200 μl 100% Ethanol and mix well    -   6. Apply to a Qiagen QIAmp DNA Blood Minikit column and spin at        6000 rpm for 1 minute    -   7. Place column in a fresh collection tube and add 500 μl of        Qiagen buffer AW1 then spin at full speed for 1 minute    -   8. Place column in a fresh collection tube and add 500 μl of        Qiagen buffer AW2 then spin at full speed for 1 minute    -   9. Empty collection tube and re-spin column at full speed for 2        minutes to dry    -   10. Add 100 μl of Qiagen buffer EB (pre-warmed to 56° C.), allow        to sit for at least 1 minute and spin at full speed for 1 minute    -   11. Add eluate back onto the column, allow to sit for at least 1        minute and spin at full speed for 1 minute    -   12. Quantitate DNA by reading OD260 on an Eppendorf        Biophotometer with a programmed factor of 1 OD260=50 μg/ml dsDNA    -   13. Store eluted DNA at −20° C.

Amplification of HLA-A Specific Sequences

HLA-A specific sequences were amplified from cell-derived genomic DNA orplasma-derived DNA according to the following protocol.

-   -   1. Amplification reactions were carried out in a total volume of        25 μl    -   2. Each reaction contained the following components        -   1 ng of cellular-derived genomic DNA or 10-20 μl of            plasma-derived DNA        -   12.5 pmoles of each primer        -   1× PCR buffer (Invitrogen) i.e: 20 mM Tris-HCl (pH 8.4), 50            mM KCl, 1.0 mM MgCl₂ (final concentrations)        -   200 μM dNTPs        -   HotStar Taq (0.625 units) (Qiagen GmbH, Germany)    -   3. Cycling was performed on a Corbett Research Palm cycler with        an initial denaturation step of 95° C. for 15 minutes followed        by 45 cycles of 95° C. for 20 seconds, 62° C. or 66° C. (see 4.)        for 30 seconds and 72° C. for 20 seconds. A final extension step        was carried out at 72° C. for 2 minutes before cooling to        ambient temperature.    -   4. Reactions targeting HLA-A A1, A2 and A3 were carried out with        an annealing temperature of 66° C. All other HLA targeted        reactions had an annealing temperature of 62° C.

Analysis of Amplification Products

Following thermo-cycling, a 10 μl aliquot from each of the reactions wasanalysed by electrophoresis through a 2.5% agarose gel in 0.5× TBEbuffer. The gel was stained with a DNA-binding fluorescent dye, andresults visualised by transillumination and photographed with a CCDcamera.

Product sizes were, compared to a 100 bp Molecular Ruler (Biorad,Hercules, Calif., USA) to determine if the correct sized product wasformed.

HLA-A Testing of Maternal Cell-Derived DNA by Commercial Methods

All maternal blood cell samples (and a single paternal sample) wereHLA-A tested using at least one commercially available product andprotocol. Two products were available; the Olerup SSP™ HLA-A LowResolution kit (Olerup SSP AB, Sweden) and LifeMATCH™ DNA typing kitwith Quick-Type™ software, version 27, (Orchid Diagnostics, Stamford,Conn.), analysed by a Luminex multiplex bead analyser (Luminex, Austin,Tex.). In each case reactions were carried out according to themanufacturers instructions.

Design and Testing of HLA-Type Specific PCR Primers

Oligonucleotide primer pairs were designed for the individual PCRamplification of HLA-A type-specific sequences (Table 1). Primers weredesigned manually using an alignment of all described HLA-A sequencesobtained from the IMGT/HLA database (http://www.ebi.ac.uk/imgt/hla).

Primers were tested for optimal annealing temperature and specificityusing an annealing gradient in PCR. This involved testing each primerpair against a template containing the specific HLA-A type targeted and,in a separate reaction, a template containing a non-specific HLA-A type.An annealing temperature for specific type reaction was chosen when astrong product was observed for the template containing the specifictype and no product was observed for the template containing thenon-specific type. Initial gradient reactions were carried out in afinal concentration of 1.5 mM MgCl₂. Once a suitable annealingtemperature had been chosen primer pair specificity was furtheroptimised by altering MgCl₂ concentrations at which point 1 mM MgCl₂ wasdetermined as the most suitable concentration to be used will all primersets.

The primers identified and tested for the examples fell into two groupswith identical optimal PCR reaction conditions, so that they can also beused in multiplex reactions, permitting the search of fetal-specific HLAsequences in only 2 reactions, followed by amplicon identification onthe basis of different amplicon lengths. (This procedure necessitatesthe use of labelled primers). Further primer design and testing willmake it possible to search for most HLA sequences using a singlemultiplex reaction.

Results and Discussion

The results of screening experiments, designed to identify fetal HLA-Atype from maternal plasma, are shown in FIGS. 1 and 2. In each case, thematernal HLA-A type was initially determined by a commercially availablemethod (Olerup or Luminex). Then a panel of eight HLA-A type-specificprimer pairs was used to screen both maternal cell-derived DNA andplasma-derived DNA. This panel included HLA-A types A01, A02, A03, A11,A24, A26, A29 and A32, giving population coverage of approximately 84%(Table 1).

TABLE 1Oligonucleotide primer sequences used in the detection of specificHLA-A types. The population frequency figures are as reported in http://ashi-hla.org/publicationfiles/archives/prepr/mori_gf.htm. HLA-Expected A type Population Oligonucleotide Oligonucleotide producttargeted frequency name sequence size A1 15.18 A1Fcv1TGTATGGCTGCGACGTGGGGC 221 (SEQ ID NO: 1) exon3 A1Rv3s2CAGGTATCTGCGGAGCCCG (SEQ ID NO: 2) A2 28.65 A2Fv3s2 GGAGCCCCGCTTCATCGCA188 (SEQ ID NO: 3) exon2 A2Rv3s2 CGCAGGGTCCCCAGGTCCA (SEQ ID NO: 4) A313.39 A3Fv2s ATGGCTGCGACGTGGGGT 2-3 (SEQ ID NO: 5) exon3 A3Rv3s2CCACTCCACGCACGTGCCA (SEQ ID NO: 6) A11 6.17 A1Fcv3s2 TATGGCTGCGACGTGGGGC188 (SEQ ID NO: 7) Exon3 A11Rv2s CCTCCAGGTAGGCTCTCT (SEQ ID NO: 8) A249.32 A24Fv3s2 ACACCCTCCAGATGATGTT 204 (SEQ ID NO: 9) exon3 A24Rv3s2CCCTCCAGGTAGGCTCTCT (SEQ ID NO: 10) A26 3.88 A26Fcv1TCCATGAGGTATTTCTACACC 216 (SEQ ID NO: 11) exon2 A26Rv2sGCAGGGTCCCCAGGTTCG (SEQ ID NO: 12) A29 3.58 A29Fcv1CCCACTCCATGAGGTATTTCA 138 (SEQ ID NO: 13) exon2 A29Rv3s2CTCCTGCTCTATCCACGGT (SEQ ID NO: 14) A32 3.70 A32Fcv1CCACTCCATGAGGTATTTCTT 233 (SEQ ID NO: 15) exon2 A25Rcv2sGTAGCGGACCGCGATCCG (SEQ ID NO: 16)

Although the maternal HLA-A type had been determined using commerciallyavailable methods the cellular-derived DNA was included to test for anynon-specific amplification. (Maternal blood contains only less than 1fetal cell per 1 million maternal cells, so that the contamination ofmaternal DNA with fetal DNA in cell-derived DNA is too small to resultin a fetal-specific signal. This is in contrast to cell-free plasma DNAwhich contains typically >5% fetal DNA). An example of such non-specificamplification can be seen in sample 33 (FIG. 1), where the primer pairdirected at A03 is non-specifically amplifying a target in the maternalA02/A30 sample. As the A03 product appears in both the cellular andplasma-derived DNA samples it can be excluded as a fetal-specific type.The plasma-derived DNA from sample 59 also shows a non-specific productproduced by the A03 primer pair. This product is weak and we have notedoccasional cross-reactivity with the A03 primer pairs in the past (asnoted above for sample 33). Regardless, HLA-A03, and other, allelespecific primers which do not result in any non-specific products canreadily be designed and tested using routine techniques well within thecapacity of the skilled addressee.

Each of the examples presented in FIG. 1 shows a clear, strong band thatis unique to the plasma-derived DNA sample and thus is predicted to beof fetal origin. For sample 33 the unique foetal signal is HLA-A01, forsample 59 the unique fetal signal is HLA-A24 and for sample 69 theunique foetal signal is HLA-A29.

Paternal DNA was available in only one sample. The knowledge of paternalHLA-A type allowed us to confirm the results by specifically targetingthese types in the maternal plasma-derived DNA sample and the results ofthis experiment are shown in FIG. 2. It can be seen in panels A & B thatthe primer panel is correctly predicting maternal and paternal HLA-Atype. Panel C shows the targeted reactions and included maternal,paternal and plasma-derived DNA. A single, strong, HLA-A29 signal isvisible in the plasma sample indicating that this allele has beeninherited by the foetus from the father.

Example 2 Prophetic

A lymphocyte preparation is obtained form a pregnant female and HLAtyped using HLA serological typing trays obtained from Pel-FreezClinical Systems, LLC (Wisconsin, USA).

A list of HLA alleles which the mother does not possess is prepared.Preferably, the list is ordered to ensure that the more common HLAalleles are tested first. This will reduce costs, as less HLA allelesshould need to be screened before a HLA antigen specific to the fetalcells is identified.

A maternal plasma sample comprising fetal DNA is prepared as outlined inWO 98/39474. The sample comprising fetal DNA is HLA typed for allelesnot present in the mother using multiple sets of PCR primers designed toamplify specific HLA alleles followed by the detection of amplificationproducts. Positive reactions (amplification products) will identify HLAalleles present in the fetal DNA but absent in the maternal DNA whichcan be used as fetal DNA/cell markers.

If commercial SSP kits are utilized (for instance using Olerup SSP™ HLAproducts, Qiagen AS, Norway) the reaction products will include maternalamplified alleles. However, since the MA type of the mother has beendetermined the relevant amplification products can be discounted asnon-fetal specific amplification products.

Example 3 Prophetic Fetal DNA Quantitation Procedure Using HLAType-Specific Standard Curve

Stocks of HLA-type specific DNA samples are prepared from genomic DNA ofblood donors, and their exact DNA quantity is determined by standard(spectrophotometric or chemical) methods. The stocks are stored assingle-use aliquots.

In the instance where fetal HLA typing from maternal plasma using amethod of the invention has revealed HLA-A29 to be a unique fetal alleleof the fetus a 1:3 dilution series from a stored aliquot of HLA-A29standard DNA is made, covering a range from 5 ng to 1 pg DNA (=standardcurve). A PCR primer pair is chosen that is specific for HLA-A29, forexample; Forward CCCACTCCATGAGGTATTTCA (SEQ ID NO:13) and ReverseCTCCTGCTCTATCCACGGT (SEQ ID NO:14). Real-time quantitative PCR reactionsare set up, using the standard curve preparation as well as 3-5replicates of an aliquot (5-40 ul) of the DNA prepared from the maternalplasma (=test samples).

Reactions are carried out in a real-time Q-PCR apparatus (for example,Stratagene Mx3000P quantitative thermal cycler), using optimisedconditions as determined for the HLA-A29 primer pair used. For example,the reaction may comprise (final concentration) 12.5 pmoles of eachprimer, 1× PCR buffer (Invitrogen) i.e: 20 mM Tris-HCl (pH 8.4), 50 mMKCl, 1 to 6 mM MgCl₂, 0.01 to 1.0 μM molecular beacon or for SYBR Greendetection 0.05 to 2 times the supplied concentration (#S-9430, Sigma,USA), 200 μM dNTPs, HotStar Tag (0.625 units) (Qiagen GmbH, Germany).Cycling is performed with an initial denaturation step of 95° C. for 15minutes followed by 50 cycles of 95° C. for 20 seconds, 62° C. for 30seconds and 72° C. for 20 seconds. A final extension step is carried outat 72° C. for 2 minutes before cooling to ambient temperature. In thecase of SYBR detection a denaturation curve is generated by the Mx3000Pinstrument in order to validate the product formed.

The accumulation of PCR product is measured by SYBR green or anyhybridisation-based dye (beacon) system designed for the targetedamplicon. An example of a suitable molecular beacon for detectingamplification of the HLA-A29 allele isGCTCGGGTGACGACACGCAGTTCGTGCGGACCGAGC (SEQ ID NO:17) which is labelledwith a 5′ fluorophore—HEX and a 3′ quencher—BHQ1. The numbers of PCRcycles needed to reach a set amount of PCR product (signal threshold) isdetermined (=Ct). Ct is a function of the number of target sequencesinitially present in the samples. The processing by the real-time Q-PCRapparatus relates the Ct from the test samples to the Ct from thestandard curve (with known DNA quantities), to yield the quantity ofHLA-A29 DNA in the test samples. The concentration of fetal DNA(HLA-A29) in the maternal plasma sample is determined by calculatingback through the dilution factors to the amount of sample used.

Example 4 Prophetic Fetal DNA Quantitation Using a Universal Primer forthe Standard Curve

This procedure is in essence as the one outlined above in Example 3,except that the standard curve is based on the PCR amplification of anon-polymorphic sequence (=universal standard), thus utilising the sameDNA stock for all standard curves, instead of one DNA stock for each HLAtype. To correct for differences in the PCR amplification efficiencies,a set of “relative efficiency factors” is determined experimentally,relating amplification efficiency for the universal standard to theefficiency of each of the HLA-type specific amplifications with theoptimised primers and conditions.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

All publications discussed above are incorporated herein in theirentirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

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1.-43. (canceled)
 44. A method of identifying an allele of a fetal cell,said allele not being present in maternal cells, the method comprising;i) identifying alleles which are not present in maternal cells, ii)obtaining a sample from the mother comprising fetal and maternal nucleicacids, and iii) screening nucleic acids from the sample for at least oneallele not present in the maternal cells.
 45. A method of identifying anallele of a fetal cell, said allele not being present in maternal cells,the method comprising; i) obtaining a sample from the mother comprisingfetal and maternal nucleic acids, ii) typing at least one locus from thesample obtained in step i), iii) comparing the alleles identified instep ii) with alleles of the same locus possessed by the mother, and iv)selecting an allele typed in step iii) which is not possessed by themother.
 46. The method of claim 44, wherein the father is not typed forany alleles.
 47. The method of claim 44, wherein the sample is obtainedduring the first trimester of pregnancy.
 48. The method of claim 44,wherein if the mother has not been typed for an allele before the methodis performed the method further comprises typing alleles of at least onelocus from maternal cells.
 49. The method of claim 48, wherein thetyping is performed by obtaining a genomic DNA sample from the motherand exposing the DNA to amplification and/or sequencing procedures. 50.The method of claim 48, wherein the typing is performed by usingantibodies which bind the protein products of specific alleles.
 51. Themethod of claim 48, wherein typing alleles of at least one locus frommaternal cells is performed on nucleated blood cells, saliva or hairfollicles.
 52. The method of claim 44, wherein the father is typed foralleles at the same locus/loci as the mother, and alleles are identifiedwhich are not present in the mother but which are present in the father.53. The method of claim 44, wherein the allele is from an HLA locus. 54.The method of claim 44, wherein the sample is derived from plasma, serumor urine.
 55. The method of claim 44, wherein the sample is subjected toaffinity chromatography to enrich nucleic acids from the sample.
 56. Themethod of claim 44, wherein numerous potential alleles at a particularlocus are investigated in the sample simultaneously by a multiplexamplification procedure.
 57. The method of claim 44, wherein nucleicacid in the sample is DNA or RNA.
 58. The method of claim 44, whereinthe mother is a mammal.
 59. The method of claim 58, wherein the mammalis a human.
 60. A method of quantifying fetal specific nucleic acids ina sample comprising fetal and maternal nucleic acids, the methodcomprising identifying an allele which is present in the DNA of a fetalcell but absent from maternal DNA using a method of claim 1, anddetermining the concentration of the allele in the sample.
 61. Themethod of claim 60, wherein the allele is quantified by exposing thesample to a nucleic amplification procedure which specifically amplifiesthe allele, and detecting the amplification product.
 62. The method ofclaim 61, wherein the allele is an HLA allele.